Mosquito Saliva in Dengue Virus Pathogenesis. Dengue virus is a single-stranded RNA Flavivirus that causes dengue fever (DF), the most prevalent arthropod-borne viral illness in humans. Dengue viruses cause an estimated 5 million new cases of DF and 250,000 cases of dengue hemorrhagic fever (DHF) per year in tropical and subtropical areas of the world, with >100 countries with endemic transmission. Currently, no specific treatment or licensed vaccine is available for either DF or DHF. Given its global burden, increased travel and military activity in dengue-endemic areas, there is an urgent need for animal models of disease, to understand the basics of dengue pathogenesis, especially the mechanisms that lead to the more severe DHF, and whether vaccination might actually enhance disease. This is the first revision of a 5-year grant proposal to study the effect of mosquito salia on dengue virus pathogenesis. We have recently established a mouse model of dengue fever, using humanized mice (transplanted with human immune system stem cells), and we infect these mice by mosquito bite. Our preliminary data point to a major enhancement of dengue virus replication and pathogenesis in these mice, due to mosquito saliva components; the mice develop the typical saddleback, prolonged viremia and rash that occurs in humans, before going on to develop severe disease, or DHF. These mice also develop human antibodies to dengue after mosquito bite, in contrast with no antibodies after virus injection by syringe, providing evidence that these mice can mount an adaptive immune response. These results have led us to propose studies to define the specific mechanisms of infection and pathogenesis in this unique laboratory system that mimics the natural cycle of dengue transmission. The specific aims are: 1). Define dengue virus pathogenicity in mosquito-bitten, humanized mice. Definition of determinants of dengue pathogenesis in humanized mice, including pinpointing specific genome regions involved in viral replication and tropism, measurement of human antibodies and cellular immunity after mosquito infection, and the possibility of inducing typical DHF, with hemorrhagic signs of disease, after serial infection; and 2). Measure the effect of specific saliva components on immune response in humanized mice. Evaluation of the individual and combined effects of mosquito saliva components on rates of infection and dengue pathogenesis in humanized mice, including using injection of recombinant saliva proteins (9 total) and overexpression of some (4 max.) of these saliva proteins, in engineered mosquitoes. Dengue is a category A Priority Pathogen in the NIAID Biodefense Research Program; the results of this project would benefit many other arbovirus and parasite studies, especially those that concern the role of arthropod saliva in disease progression.